Main Procedure
1. Retrieve flash frozen half kidney from the -80°C storage. While frozen, use tweezers or scissors to remove the kideny and place it in a pre-chilled Sample Dissociation Tube.
2. Add 200 µl of the Lysis Buffer and wait for the kidney to mostly thaw. Using sterile scissors, chop up the kidney into small pieces, closing and opening scissors at least 50 times.
3. Then using the green pestle provided in the room-temperature pack, grind up the tissue, pressing down and twisting at least 20 times or until the solution is smooth with no visible chunks.
4. Next, add 300 µl of the Lysis buffer and pipette up and down, making sure that there are no chunks that block the pipette tip. Incubate the solution on ice for 8 minutes, starting the timer after completing the grinding step.
5. Next, pipette the dissociated tissue solution onto the Nuclei Isolation Column in the collection Tube (both included in room temperature Consumables) and spin in the centrifuge for 16,000 rcf, 20 sec, at 4°C. Make sure that this cycle is evenly balanced in the centrifuge, because of the high speed.
6. After the cycle ends, discard the column and vortex the flow-through for 10 seconds to re-suspend the nuclei. Then spin in the centrifuge at 700 rcf, 3 min, 4°C. Make sure that when placing the Nuclei Isolation Column, position it within the centrifuge so that the hinge of the lid points out, the front tab facing in. This will make it easier to find the pellet and avoid losing nuclei.
7. Next, remove the supernatant, being careful to avoid the pellet and angling the pipette tip away from the hinge side of the column.
8. Re-suspend the pellet with 500 µl of the Debris Removal Buffer. Spin at 700 rcf for 10 minutes at 4°C. While waiting, mix the final Resuspension Buffer, which should be 0.04% BSA in PBS.
9. Remove the supernatant again and resuspend in 1 mL of the Wash and Resuspension Buffer. Spin at 500 rcf for 5 minutes, 4 °Cs C. Remove supernatant, resuspend again in 1 mL of Wash and Resuspension Buffer. Spin again at 500 rcf for 5 minutes, 4°C.
10. Finally, resuspend the. nuclei pellet in 500 µl of the Final Wash and Resuspension Buffer (0.04% BSA). Vortex the solution for 3 seconds and let it sit undisturbed for 15 minutes.
Now, depending on QC or following uses, use the concentrated solution.